The broad long-term objective is to obtain new information about the metabolic signals for insulin secretion. The immediate purpose of this project is to gain a better understanding of the role of mitochondrial biosynthetic pathways in insulin secretion. Glucose, the most potent insulin secretagogue, and all other fuel secretagogues stimulate insulin secretion via their metabolism in mitochondria. Our work and that of others has implicated the biosynthesis (anaplerosis) of citric acid cycle intermediates and their export from mitochondria to the cytosol (cataplerosis) in signaling insulin secretion. Aim 1 is to study the potential products of anaplerosis and will test the hypothesis that the metabolism of all fuel insulin secretagogues shares final common pathways involving mitochondria and that discerning these pathways will yield new clues about mitochondrial factors that couple metabolism and exocytosis. We will follow up a recent surprising discovery from our laboratory that citrate oscillates in beta cell mitochondria supplied with pyruvate and in intact beta cells supplied with glucose, it is hypothesized that citrate can coordinate mitochondrial metabolism with overall cellular metabolism. Studies are planned to determine whether citric acid cycle intermediates besides citrate oscillate in intact ceils, whether non-glucose secretagogues cause oscillations in citric acid cycle intermediates, to learn how mitochondria become synchronized to produce citrate oscillations, and to investigate the regulation of citrate oscillations. Aim 2 will focus on certain extramitochondrial actions of mitochonddal products to test the hypothesis that most citric acid cycle intermediates have signaling roles in insulin secretion. In Aim 2A small interfering RNAs will be used to specifically lower levels of certain enzymes that use mitochondrial products. Resulting decreases in insulin release will be compared with alterations in metabolic pathways. In Aim 2B limited focused studies of beta ceil cataplerosis in type 2 diabetes will be performed.